Theiler received his medical training at St. Thomas’s Hospital, London, and the London School of Hygiene and Tropical Medicine, graduating in 1922. In that year he joined the department of tropical medicine at Harvard Medical School, Boston. There he carried out important studies of amebic dysentery and rat-bite fever and began work on yellow fever.

In 1930 Theiler joined the Rockefeller Institute (now Rockefeller University) in New York City, where he continued his research on infectious diseases, including yellow fever. With the discovery in 1928 that rhesus monkeys were susceptible to the virus responsible for yellow fever, researchers began to develop vaccines against the disease. Theiler discovered that the common mouse is also susceptible to the yellow fever virus, a finding that facilitated the vaccine research. In the late 1930s Theiler developed the first attenuated, or weakened, strain of the virus. Further studies led to the development of the improved 17D strain that became widely used for human immunization against yellow fever.